Preparation method of tumor-enhanced tumor-infiltrating lymphocytes
A lymphocyte and tumor infiltration technology, which is applied in the field of tumor infiltrating lymphocyte preparation, can solve the problems of short survival time, immune cells do not have tumor killing function, and TILs of different quality, so as to accelerate clearance, improve tumor killing activity, and improve The effect of lethality
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Embodiment 1
[0074] The patient, male, 66 years old, the prostate tumor tissue was fully washed with normal saline, and the normal tissue and necrotic area around the prostate cancer specimen were removed. Put the prostate cancer specimen in a sterile petri dish, add a small amount of normal saline, and use sterilized surgical scissors to cut out a size of about 1-8mm from different areas around the specimen 3 The number of small tissue blocks is generally not less than 8. A small piece of prostate cancer tissue was taken and placed in a well of a 24-well plate, each well containing 2ml of X-VIVO15 medium, IL-7 (10ng / mL), IL-15 (20ng / mL), IL- 21 (20ng / mL), 1% double antibody, 5% human serum albumin, β-mercaptoethanol (55uM) and hepes (10mM), and other wells were operated in the same way. Place the 24-well plate at 37°C, 5% CO 2 cultured in a cell culture incubator. The proliferation of lymphocytes around the tissue blocks in each well was observed microscopically every other day. Regar...
Embodiment 2
[0076] The patient, male, 58 years old, the enhanced TILs culture method was the same as the above method (harvested cells 78×10 8 indivual).
Embodiment 3
[0078] The patient, male, 74 years old, the enhanced TILs culture method was the same as the above method (harvested cells 46×10 8 indivual).
[0079] like figure 2 As shown, General Biosystems (Anhui) Co., Ltd. was commissioned to construct the PBR and IL-15 super complex, according to the extracellular region of PD1 (SEQ ID NO: 1), the intracellular region of 41BB (SEQ ID NO: 2), △ Truncation (SEQ ID NO: 3) of IL2RB (YRHQ), P2A linking peptide (SEQ ID NO: 21), signal peptide (SEQ ID NO: 7), "suicide gene" R structure (SEQ ID NO: 8), Linker1 (SEQ ID NO: 9), Interleukin-15 mutant IL-15-M-N72D (SEQ ID NO: 10), Linker2 (SEQ ID NO: 11), Sushi structure of interleukin-15 receptor alpha The sequence of the domain (SEQ ID NO: 12), CD-4 transmembrane region (SEQ ID NO: 13) was assembled. After optimization, the gene is routinely synthesized and cloned into the vector plasmid vector.
[0080] 所述的PD1 DNA序列ATCCCAATGCAGCAGGCGCCCTGGCCAGTCGTCTGGGCGGTGCTACAACTGGGCTGGCGGCCAGGATGGTTCTTAG...
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